RING1B-BMI1f was diluted to 1 μM final concentration in EMSA buffer (50 mM HEPES, pH 7.2, 100 mM NaCl, 10 mM MgCl2, 1 μM ZnCl2, 1 mM DTT, 5% glycerol, 0.01% BSA) and incubated with varying concentrations of compound at room temperature for 90 minutes. H2BOG488 nucleosomes were added to a 10 ng/μL final concentration in 10 μL reaction and reactions were incubated at room temperature for 30 minutes. 1 μL DNA loading dye was added to each reaction (Life Technologies). Reaction species were separated by electrophoresis at 4° C under native conditions using 7.5% Tris-glycine acrylamide gel (BioRad). Gels were imaged with Typhoon Trio+ 9410 Variable Mode Imager (GE Healthcare) and analyzed with ImageQuant TL 7.0 (GE Healthcare).
Tris glycine acrylamide gel
Manufactured by Bio-Rad
The 7.5% Tris-glycine acrylamide gel is a laboratory equipment used for the separation and analysis of proteins in electrophoresis experiments. It is a pre-cast polyacrylamide gel with a 7.5% concentration of acrylamide, a common matrix material used in gel electrophoresis.
Automatically generated - may contain errors
2 protocols using tris glycine acrylamide gel
1
RING1B-BMI1 Nucleosome Binding Assay
2
RING1B-BMI1 Nucleosome Binding Assay
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RING1B-BMI1f was diluted to 1 μM final concentration in EMSA buffer (50 mM HEPES, pH 7.2, 100 mM NaCl, 10 mM MgCl2, 1 μM ZnCl2, 1 mM DTT, 5% glycerol, 0.01% BSA) and incubated with varying concentrations of compound at room temperature for 90 minutes. H2BOG488 nucleosomes were added to a 10 ng/μL final concentration in 10 μL reaction and reactions were incubated at room temperature for 30 minutes. 1 μL DNA loading dye was added to each reaction (Life Technologies). Reaction species were separated by electrophoresis at 4° C under native conditions using 7.5% Tris-glycine acrylamide gel (BioRad). Gels were imaged with Typhoon Trio+ 9410 Variable Mode Imager (GE Healthcare) and analyzed with ImageQuant TL 7.0 (GE Healthcare).
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